Super-Resolution Microscopy and Single-Molecule Tracking Reveal Distinct Adaptive Dynamics of MreB and of Cell Wall-Synthesis Enzymes

Abstract

The movement of filamentous, actin-like MreB and of enzymes synthesizing the bacterial cell wall has been proposed to be highly coordinated. We have investigated the motion of MreB and of RodA and PbpH cell wall synthesis enzymes at 500 ms and at 20 ms time scales, allowing us to compare the motion of entire MreB filaments as well as of single molecules with that of the two synthesis proteins. While all three proteins formed assemblies that move with very similar trajectory orientation and with similar velocities, their trajectory lengths differed considerably, with PbpH showing shortest and MreB longest trajectories. These experiments suggest different on/off rates for RodA and PbpH at the putative peptidoglycan-extending machinery (PGEM), and during interaction with MreB filaments. Single molecule tracking revealed distinct slow-moving and freely diffusing populations of PbpH and RodA, indicating that they change between free diffusion and slow motion, indicating a dynamic interaction with the PGEM complex. Dynamics of MreB molecules and the orientation and speed of filaments changed markedly after induction of salt stress, while there was little change for RodA and PbpH single molecule dynamics. During the stress adaptation phase, cells continued to grow and extended the cell wall, while MreB formed fewer and more static filaments. Our results show that cell wall synthesis during stress adaptation occurs in a mode involving adaptation of MreB dynamics, and indicate that Bacillus subtilis cell wall extension involves an interplay of enzymes with distinct binding kinetics to sites of active synthesis.

Single molecule/particle tracking analysis program SMTracker 2.0 reveals different dynamics of proteins within the RNA degradosome complex inBacillus subtilis

“…Up to 25% of the intensity was used via a YFP filter set (BrightLine 500/24, Beamsplitter 520 and BrightLine 542/27), generating a laser power density of about 160 W cm –2 in the object plane. We have previously shown that cells continue to grow following exposure to this intensity, following the 2 min-long experiments ( 15 ). NIS-elements was used for image acquisition.…”

“…To further investigate different modes of diffusion, we have included MSD-based analyses of tracks to determine if Brownian motion, sub- or superdiffusion may underlay molecule movement, which we have e.g. employed to analyse motion of actin-like MreB protein at a single molecule level ( 15 ). A further handy tool we have developed is the ability to study proximity of proteins and defined subcellular sites.…”

Dynamics of Bacterial Signal Recognition Particle at a Single Molecule Level

“…However, data on single molecule dynamics are scarce, although they can be highly powerful to describe spatiotemporal aspects of processes within live cells. In this study, we have visualized the movement of SRP, FtsY, and of ribosomes in Shewanella putrefaciens CN32, henceforth called S. putrefaciens, with a localization precision of below 50 nm (Dersch et al, 2020). We were able to generate functional fluorescent protein fusions to Ffh, FtsY, and the ribosomal large subunit protein L1, which are inserted at the original gene locus and are driven by the original promoter, generating fusions expressed at physiological levels.…”

Elongation at Midcell in Preparation of Cell Division Requires FtsZ, but Not MreB nor PBP2 in Caulobacter crescentus

“…It is clear that MreB is a crucial part of the elongasome, but its exact role remains puzzling (Ducret and Grangeasse, 2021). Instead of a scaffold that actively directs the movement of all other elongasome proteins, MreB seems to have a subtler coordinating role (Dion et al, 2019;Dersch et al, 2020), in which its movement in part seems to depend on PG synthases that take the lead (Dominguez-Escobar et al, 2011;Garner et al, 2011;van Teeffelen et al, 2011;Özbaykal et al, 2020).…”